Summary Despite having reduced malaria transmission to historically low level, in many Asia-Pacific countries endemic areas transmission continues and common vector control tools seem not able to interrupt transmission. The reasons for the resilience of malaria transmission are on unclear but are likely to involve not only parasite but also host and vector factors. At low transmission, asymptomatic infections account for up to 90% of infections and it they are able to transmit efficiently they could be a key component of the infectious reservoir. The only way to accurately ascertain if an infection can transmit is to feed the blood to mosquitoes and determine rate and density of infections of the mosquito midgut. Such xenodiagnostic studies are severely lacking, in particular in predominantly P. vivax endemic area. We are now proposing to conduct an in-depth dissection of the contribution of host, parasite and vector factors to maintaining malaria transmission by conducting a set studies that combine assessment of the infectivity of well characterized field samples using direct membrane feeding (DFMA) with in-depth laboratory studies in 3 sites with substantially different transmission scenarios. Initially we will investigate the relative infectivity of asymptomatic and symptomatic infections establish the association of parasite and gametocytes density with infectivity and study effect of transmission blocking immunity, vector microbiome and type parasite for gene associated with evasion of mosquito immune responses and markers of artemisinin and partner drugs resistance to determine their effect on transmission potential. As parasite and gametocyte fluctuate over time, so may their infectivity. We will therefore determine the temporal variation in infectivity an in-depth cohort study in PNG, where asymptomatic carriers will be followed for 3 weeks and mosquitoes fed every 3-4 days. Addition, we will determine proportion of pre- and post-treatment infectivity in in artemisinin-resistant and sensitive symptomatic P. falciparum. Together with our large epidemiological datasets (see Project 1), this will allow us not only clarify the contribution of asymptomatic infections to transmission but also resistance may come with a transmission associated fitness cost, which may slow down the spread of resistance. Both human and vector behavior may limit human-vector contact in the Asia-Pacific, where vectors are frequently / predominantly outdoor biting, leading to transmission away from people?s dwelling. The low biting rate and unclear location of human-vector contact makes it difficult to identify its determinants. We will we study patterns of human vector contact by determining the presence of antibodies against salivary gland protein antibodies proteins from An. dirus. An. minimus and An. Farauti in samples from cross-sectional and cohort studies in Project 1. By providing an in-depth understanding of the transmission characteristics at each of our sites, the proposed studies will not only contribute to developing biologically accurate transmission models but will help us design better methods to track and eliminate transmission even in hard to track populations such asymptomatic carriers.